JPWO2019217803A5 - - Google Patents

Description

Various embodiments described herein include administering to a subject the modified human host cells described herein to reduce the size of the tumor or reduce the rate of tumor growth in the subject. Provide a way to make it.
[Invention 1001]
(a) One or more nucleotide sequences and homologous to a fragment of the auxotrophic lotus or a complement to the auxotrophic lotus.
(b) Transgene encoding a therapeutic factor optionally linked to an expression control sequence
Donor template including.
[Invention 1002]
A single-stranded donor template for the invention 1001.
[Invention 1003]
A double-stranded donor template for the invention 1001.
[Invention 1004]
A donor template of the invention 1001, which is a plasmid or DNA fragment or vector.
[Invention 1005]
The donor template of the invention 1004, which is a plasmid containing the elements required for replication, optionally including a promoter and 3'UTR.
[Invention 1006]
(a) One or more nucleotide sequences and homologous to the fragment of the auxotrophic inducible lous or to the complement of the auxotrophic inducible lotus.
(b) Transgene encoding a therapeutic factor
Vector containing.
[Invention 1007]
The vector of the present invention 1006, which is a viral vector.
[Invention 1008]
The viral vector of the present invention 1007 selected from the group consisting of vectors of retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus.
[Invention 1009]
The viral vector of the present invention 1007, further comprising a gene required for replication of the viral vector.
[Invention 1010]
A donor template or vector of any of the preceding inventions, wherein nucleotide sequences homologous to the auxotrophic inducing locus fragment or complement thereof are flanking on either side of the transgene.
[Invention 1011]
The donor template or vector of any of the preceding inventions, wherein the auxotrophic induction locus is a gene encoding a protein involved in the synthesis, reuse or salvage of auxotrophic factors.
[Invention 1012]
The donor template or vector of any of the preceding inventions, wherein the auxotrophic induction locus is within the gene of Table 1 or within the region controlling the expression of the gene of Table 1.
[Invention 1013]
The donor template or vector of any of the preceding inventions, wherein the auxotrophic induction locus is within a gene encoding uridine monophosphate synthase.
[Invention 1014]
The donor template or vector of any of the preceding inventions, wherein the auxotrophic induction locus is within a gene encoding holocarboxylase synthase.
[Invention 1015]
The donor template or vector of any of the preceding inventions, wherein the nucleotide sequence homologous to the fragment of the auxotrophic inducible locus is 98% identical to at least 200 contiguous nucleotides of the auxotrophic inducible locus.
[Invention 1016]
The nucleotide sequence homologous to the fragment of the auxotrophic induction locus is 98% identical to at least 200 consecutive nucleotides of either the human uridine monophosphate synthase or the holocarboxylase synthase or the gene of Table 1. Prior to any donor template or vector of the invention.
[Invention 1017]
A donor template or vector of any of the preceding inventions further comprising an expression control sequence operably linked to the transgene.
[Invention 1018]
The donor template or vector of the present invention 1017, wherein the expression control sequence is a tissue-specific expression control sequence.
[Invention 1019]
The donor template or vector of the present invention 1017, wherein the expression control sequence is a promoter or enhancer.
[Invention 1020]
The donor template or vector of the present invention 1017, wherein the expression control sequence is an inducible promoter.
[Invention 1021]
The donor template or vector of the present invention 1017, wherein the expression control sequence is a constitutive promoter.
[Invention 1022]
The donor template or vector of the present invention 1017, wherein the expression control sequence is a post-transcriptional regulatory sequence.
[Invention 1023]
The donor template or vector of the present invention 1017, wherein the expression control sequence is microRNA.
[Invention 1024]
A donor template or vector of any of the preceding inventions further comprising a marker gene.
[Invention 1025]
The donor template or vector of the invention 1024, wherein the marker gene comprises at least a fragment of NGFR or EGFR, at least a fragment of CD20 or CD19, Myc, HA, FLAG, GFP, or an antibiotic resistance gene.
[Invention 1026]
The introduced genes are hormones, cytokines, chemokines, interferons, interleukins, interleukin-binding proteins, enzymes, antibodies, Fc fusion proteins, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, receptors, cells. A donor template or vector of any of the preceding inventions selected from the group consisting of surface antigens, receptor antagonists, and costimulatory factors, structural proteins, cell surface antigens, ion channels, epigenetic modifiers, or RNA editing proteins. ..
[Invention 1027]
A donor template or vector of any of the preceding inventions, wherein the transgene encodes a T cell antigen receptor.
[Invention 1028]
A donor template or vector of any of the preceding inventions, wherein the transgene encodes RNA, optionally a regulatory microRNA.
[Invention 1029]
(a) cas9 protein, and
(b) Guide RNA specific for auxotrophic induced loci
A nuclease system for targeting transgene integration into the auxotrophic induction locus, including.
[Invention 1030]
A nuclease system for targeting the integration of a transgene into an auxotrophic induction locus, comprising a meganuclease specific for the auxotrophic induction locus.
[Invention 1031]
The nuclease system of the present invention 1027, wherein the meganuclease is a ZFN or TALEN.
[Invention 1032]
The nuclease system of any of the inventions 1029-1031 further comprising the donor template or vector of any of the inventions 1001-1028.
[Invention 1033]
A modified host cell of Exvivo comprising an introductory gene encoding an auxotrophic factor integrated into an auxotrophic locus, wherein the modified host cell is auxotrophic for the auxotrophic factor and the therapeutic factor. A modified host cell capable of expressing.
[Invention 1034]
A modified host cell of the invention 1033, which is a mammalian cell.
[Invention 1035]
A modified host cell of the invention 1033, which is a human cell.
[Invention 1036]
Embryonic stem cells, stem cells, precursor cells, pluripotent stem cells, artificial pluripotent stem (iPS) cells, somatic stem cells, differentiated cells, mesenchymal stem cells, neural stem cells, hematopoietic stem cells or hematopoietic precursor cells, adipose stem cells, keratinocytes , A modified host cell of the invention 1033 selected from the group consisting of skeletal stem cells, muscle stem cells, fibroblasts, NK cells, B cells, T cells, and peripheral blood mononuclear cells (PBMC).
[Invention 1037]
The modified host cell of the present invention 1033, which is derived from a cell from a subject treated with the modified host cell.
[Invention 1038]
A method for producing modified mammalian host cells.
(a) Nucleic acids encoding one or more nuclease systems that target and cleave the DNA in the nutritionally demanding induction locus, or one or more components of the one or more nuclease system. Introducing into the mammalian host cell, and
(b) Donor template or vector according to any one of the present inventions 1001 to 1028.
The method described above.
[Invention 1039]
A second nuclease or a second guide RNA for targeting and cleavage of the DNA at the second genomic locus, or a nucleic acid encoding the second nuclease or the second guide RNA, and optionally.
(b) Second donor template or vector
The method of the present invention 1038, further comprising the introduction of.
[Invention 1040]
In Exvivo, a method of targeting the integration of a transgene into a feeding-requiring induction locus of a mammalian cell, contacting the mammalian cell with any of the donor templates or vectors and nucleases of the present invention 1001-1028. Included, said method.
[Invention 1041]
The method of any of the present inventions 1038-1040, wherein the nuclease is a ZFN.
[Invention 1042]
The method of any of the present inventions 1038-1040, wherein the nuclease is TALEN.
[Invention 1043]
A method for producing modified mammalian host cells.
(a) Cas9 polypeptide or nucleic acid encoding the Cas9 polypeptide,
(b) Guide RNA specific for the auxotrophic induction locus, or nucleic acid encoding the guide RNA, and
(c) Donor template or vector from any of 1001 to 1028 of the present invention.
The method comprising introducing into the mammalian host cell.
[Invention 1044]
(a) A second guide RNA specific for the second auxotrophic induction locus, or a nucleic acid encoding the guide RNA, and optionally.
(b) Second donor template or vector
The method of 1042 of the present invention, further comprising introducing into the mammalian host cell.
[Invention 1045]
A method of targeting the integration of a transgene into a feeding-requiring induction locus of a mammalian cell in Exvivo, the donor template or vector, Cas9 polypeptide, and guide RNA of any of 1001-1028 of the present invention and the mammal. The method comprising contacting cells.
[Invention 1046]
The method of any of 1043 to 1045 of the present invention, wherein the guide RNA is a chimeric RNA.
[Invention 1047]
The method of any of 1043-1045 of the present invention, wherein the guide RNA comprises two hybridized RNAs.
[Invention 1048]
The method of any of 1038-1045 of the present invention, further comprising causing one or more single-strand breaks within the auxotrophic induction locus.
[Invention 1049]
The method of any of 1038-1045 of the present invention, further comprising causing double-strand breaks within the auxotrophic induction locus.
[Invention 1050]
The method of any of 1038-1049 of the present invention, wherein the auxotrophic induction locus is modified by homologous recombination using the donor template or vector.
[Invention 1051]
The method of any of the present inventions 1038-1049, wherein steps (a) and (b) are performed before or after expansion of the cells and optionally culture of the cells.
[Invention 1052]
(c) The method of the present invention 1051 further comprising selecting cells containing the transgene integrated into the auxotrophic induction locus.
[Invention 1053]
The above choice is
(i) Select cells that require the auxotrophy factor to survive; and optionally.
(ii) Select cells containing the transgene integrated into the auxotrophic induction locus.
The method of the present invention 1052, comprising.
[Invention 1054]
The method of the present invention 1052, wherein the auxotrophic induction locus is the gene encoding uridine monophosphate synthase and the cells are selected by contact with 5-FOA.
[Invention 1055]
A sterile composition comprising the donor template or vector of any of 1001-1028 of the invention or the nuclease system of any of 1029-1032 of the invention, and sterile water or pharmaceutically acceptable excipients.
[Invention 1056]
A sterile composition comprising the modified mammalian host cell of any of 1033-1037 of the present invention and sterile water or pharmaceutically acceptable excipients.
[Invention 1057]
Preceding A kit comprising any donor template or vector or nuclease system or modified host cell of the invention, or a combination thereof, optionally with a container or vial.
[Invention 1058]
A method of expressing a therapeutic factor in a subject,
(a) Administering any of the modified host cells of the present invention 1033-1037;
(b) Optionally, a pretreatment regimen to allow the modified cells to engraft; and
(c) Administering the auxotrophic factor
The method described above.
[Invention 1059]
The method of the present invention 1058, wherein the modified host cell and the auxotrophic factor are administered simultaneously.
[Invention 1060]
The method of the present invention 1058, wherein the modified host cell and the auxotrophic factor are administered in succession.
[Invention 1061]
The method of the invention 1058, further comprising regularly continuing administration of the auxotrophic factor for a period sufficient to promote expression of the therapeutic factor.
[Invention 1062]
The method of the present invention 1058, further comprising reducing the rate of administration of the auxotrophic factor in order to reduce the expression of the therapeutic factor.
[Invention 1063]
The method of the invention 1058, further comprising increasing the administration of the auxotrophic factor in order to increase the expression of the therapeutic factor.
[Invention 1064]
The method of the present invention 1058, further comprising discontinuing administration of the auxotrophic factor to cause a growth-inhibiting or mortal condition of the modified host cell.
[Invention 1065]
The method of the present invention 1058, further comprising temporarily discontinuing administration of the auxotrophic factor to cause a condition that results in growth inhibition of the modified host cell.
[Invention 1066]
The method of the invention 1058, further comprising continuing administration of the auxotrophic factor for a period sufficient to exert a therapeutic effect in the subject.
[Invention 1067]
The method of the present invention 1058, wherein the modified host cell is regenerative.
[Invention 1068]
The method of the invention 1058, wherein administration of the modified host cell comprises topical delivery.
[Invention 1069]
The method of the present invention 1058, wherein administration of the auxotrophic factor comprises systemic delivery.
[Invention 1070]
The method of any of the preceding inventions, further comprising obtaining said host cell from a subject to be treated prior to modification.
[Invention 1071]
A method of treating a subject with a disease, disorder, or condition in an amount sufficient to result in the expression of a therapeutic amount of the therapeutic factor: (a) the modified host cell and (b) the nutritional requirement. The method comprising administering to the subject a sex factor.
[Invention 1072]
The disease, the disorder, or the condition is cancer, Parkinson's disease, graft-versus-host disease (GvHD), autoimmune condition, hyperproliferative disorder or condition, malignant transformation, liver condition, hereditary genetic defect. The method of the present invention 1067, selected from the group consisting of genetic status, juvenile diabetes, and eye compartment status, including.
[Invention 1073]
The disease, the disorder, or the condition is selected from the group consisting of the muscular system, the skeletal system, the circulatory system, the nervous system, the lymphatic system, the respiratory system, the endocrine system, the digestive system, the excretory system, and the reproductive system. The method of the present invention 1067, which affects at least one system of the body.
[Invention 1074]
Use of a modified host cell of any of 1033-1037 of the present invention for the treatment of a disease, disorder, or condition.
[Invention 1075]
A modified host cell of any of 1033-1037 of the present invention for use in human administration or in the treatment of a disease, disorder, or condition.
[Invention 1076]
An auxotrophic factor for use in administration to a human given a modified human host cell according to any of 1033 to 1037 of the present invention.
[Invention 1077]
A method of alleviating or treating a disease or disorder in a subject in need thereof,
(a) A composition comprising a modified host cell containing a transgene encoding a protein integrated into an auxotrophic inducing locus, wherein the modified host cell is auxotrophic with respect to an auxotrophic factor; and
(b) An auxotrophic factor in an amount sufficient to result in therapeutic expression of the protein.
The method comprising administering to the subject.
[Invention 1078]
The method of the present invention 1077, wherein the auxotrophic induction locus is within a gene encoding uridine monophosphate synthase (UMPS).
[Invention 1079]
The method of the present invention 1078, wherein the auxotrophy factor is uridine.
[Invention 1080]
The method of the present invention 1077, wherein the auxotrophic induction locus is within a gene encoding holocarboxylase synthase (HLCS).
[Invention 1081]
The method of 1080 of the present invention, wherein the auxotrophy factor is biotin.
[Invention 1082]
The method of the present invention 1077, wherein the protein is an enzyme.
[Invention 1083]
The method of the present invention 1077, wherein the protein is an antibody.
[Invention 1084]
The modified host cells include embryonic stem cells, stem cells, precursor cells, pluripotent stem cells, artificial pluripotent stem (iPS) cells, somatic stem cells, differentiated cells, mesenchymal stem cells, nerve stem cells, hematopoietic stem cells or hematopoietic precursors. The method of the invention 1077, which is a cell, adipose stem cell, keratinocyte, skeletal stem cell, muscle stem cell, fibroblast, NK cell, B cell, T cell, or peripheral blood mononuclear cell (PBMC).
[Invention 1085]
The method of the present invention 1077, wherein the modified host cell is a mammalian cell.
[Invention 1086]
The method of the present invention 1085, wherein the mammalian cell is a human cell.
[Invention 1087]
The method of the present invention 1077, wherein the modified host cell is derived from the subject in which the modified host cell is treated with the modified host cell.
[Invention 1088]
The method of the present invention 1077, wherein the composition and the auxotrophic factor are administered simultaneously.
[Invention 1089]
The method of the present invention 1077, wherein the composition and the auxotrophic factor are administered sequentially.
[Invention 1090]
The method of the present invention 1089, wherein the composition is administered prior to the auxotrophic factor.
[Invention 1091]
The method of the present invention 1077, wherein the composition and the auxotrophic factor are administered simultaneously.
[Invention 1092]
The method of the invention 1077, wherein administration of the auxotrophic factor is continued periodically for a period sufficient to promote therapeutic expression of the protein.
[Invention 1093]
The method of the present invention 1077, wherein the administration of the auxotrophic factor is reduced in order to reduce the expression of the protein.
[Invention 1094]
The method of the present invention 1077, wherein the administration of the auxotrophic factor is increased in order to increase the expression of the protein.
[Invention 1095]
The method of the present invention 1077, wherein discontinuation of administration of the auxotrophic factor induces proliferation inhibition or cell death of the modified host cell.
[Invention 1096]
The method of the present invention 1077, wherein administration of the auxotrophic factor is continued for a period sufficient to exert a therapeutic effect in the subject.
[Invention 1097]
The method of the present invention 1077, wherein the modified host cell is regenerative.
[Invention 1098]
The method of the present invention 1077, wherein administration of the composition comprises topical delivery.
[Invention 1099]
The method of the present invention 1077, wherein administration of the auxotrophic factor comprises systemic delivery.
[Invention 1100]
The method of the present invention 1077, wherein the disease is lysosomal storage disease (LSD).
[Invention 1101]
The lysosome accumulation disease (LSD) is Gauche disease (type 1/2/3), MPS2 (hunter) disease, Pompe disease, Fabry disease, Clave disease, hypophosphatase disease, Niemann-Pick disease type A / B, MPS1, The method of the present invention 1100, which is MPS3A, MPS3B, MPS3C, MPS3, MPS4, MPS6, MPS7, phenylketonuria, MLD, Sandhoff's disease, Tay-Sachs' disease, or Batten's disease.
[Invention 1102]
The enzymes are glucocerebrosidase, idulsulfase, alglucosidase alpha, agarsidase alpha, agarsidase beta, galactosylceramidase, ashotase alpha, acidic sphingomyelinase, laronidase, heparan N-sulfatase, alpha-N-. Acetylglucosaminidase, heparan-α-glucosaminide N-acetyltransferase, N-acetylglucosamine 6-sulfatase, erosulfase alpha, glasulfate, B-glucuronidase, phenylalanine hydroxylase, arylsulfatase A, hexosaminidase-B, hex The method of the present invention 1082, which is sosaminidase-A, or tripeptidyl peptidase 1.
[Invention 1103]
The method of the present invention 1077, wherein the disease is Friedreich ataxia, hereditary angioedema, or spinal muscular atrophy.
[Invention 1104]
The method of the present invention 1077, wherein the protein is frataxin, a C1 esterase inhibitor, or SMN1.
[Invention 1105]
A method of reducing the size of a tumor or slowing the growth of a tumor in a subject, comprising administering to the subject a modified host cell of any of 1033-1037 of the present invention.

Claims (89)

(a) One or more nucleotide sequences and homologous to a fragment of the auxotrophic lotus or a complement to the auxotrophic lotus.
(b) A donor template containing a transgene encoding a therapeutic factor, optionally linked to an expression control sequence. The donor template according to claim 1, which is a single chain. The donor template according to claim 1, which is double-stranded. The donor template of claim 1, which is a plasmid or DNA fragment or vector. The donor template of claim 4, which is a plasmid containing the elements required for replication, optionally including a promoter and 3'UTR. (a) One or more nucleotide sequences and homologous to the fragment of the auxotrophic inducible lous or to the complement of the auxotrophic inducible lotus.
(b) A vector containing a transgene encoding a therapeutic factor. The vector according to claim 6, which is a viral vector. The viral vector according to claim 7, which is selected from the group consisting of vectors of retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. The viral vector according to claim 7, further comprising a gene required for replication of the viral vector. The donor template or vector according to any one of claims 1 to 9 , wherein a nucleotide sequence homologous to the fragment of the auxotrophic induction locus or a complement thereof is adjacent to both sides of the transgene. The donor template or vector according to any one of claims 1 to 10 , wherein the auxotrophy-inducing lous coition is a gene encoding a protein involved in the synthesis, reuse or salvage of an auxotrophy factor. The donor template or vector according to any one of claims 1 to 11 , wherein the auxotrophic induction locus is within the gene of Table 1 or within the region controlling the expression of the gene of Table 1. The donor template or vector according to any one of claims 1 to 12 , wherein the auxotrophic induction locus is in a gene encoding uridine monophosphate synthase. The donor template or vector according to any one of claims 1 to 13 , wherein the auxotrophic induction locus is in a gene encoding holocarboxylase synthase. 13 . Donor template or vector. The nucleotide sequence homologous to the fragment of the auxotrophic induction locus is 98% identical to at least 200 consecutive nucleotides of either the human uridine monophosphate synthase or the holocarboxylase synthase or the gene of Table 1. The donor template or vector according to any one of claims 1 to 15 . The donor template or vector according to any one of claims 1 to 16 , further comprising an expression control sequence functionally linked to the transgene. The donor template or vector according to claim 17, wherein the expression control sequence is a tissue-specific expression control sequence. 17. The donor template or vector of claim 17, wherein the expression control sequence is a promoter or enhancer. 17. The donor template or vector of claim 17, wherein the expression control sequence is an inducible promoter. 17. The donor template or vector of claim 17, wherein the expression control sequence is a constitutive promoter. 17. The donor template or vector according to claim 17, wherein the expression control sequence is a post-transcriptional regulatory sequence. 17. The donor template or vector according to claim 17, wherein the expression control sequence is microRNA. The donor template or vector according to any one of claims 1 to 23 , further comprising a marker gene. 24. The donor template or vector of claim 24, wherein the marker gene comprises at least a fragment of NGFR or EGFR, at least a fragment of CD20 or CD19, Myc, HA, FLAG, GFP, or an antibiotic resistance gene. The introduced genes are hormones, cytokines, chemokines, interferons, interleukins, interleukin-binding proteins, enzymes, antibodies, Fc fusion proteins, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, receptors, cells. 13. Donor template or vector. The donor template or vector according to any one of claims 1 to 26 , wherein the transgene encodes a T cell antigen receptor. The donor template or vector according to any one of claims 1 to 27 , wherein the transgene encodes RNA, optionally a regulatory microRNA. (a) cas9 protein, and
(b) Guide RNA specific for auxotrophic induction loci
A nuclease system for targeting transgene integration into the auxotrophic induction locus, including. A nuclease system for targeting the integration of a transgene into an auxotrophic induction locus, comprising a meganuclease specific for the auxotrophic induction locus. 30. The nuclease system of claim 30 , wherein the meganuclease is a ZFN or TALEN. The nuclease system of any one of claims 29-31, further comprising the donor template or vector of any one of claims 1-28. A modified host cell of Exvivo comprising an introductory gene encoding an auxotrophic factor integrated into an auxotrophic locus, wherein the modified host cell is auxotrophic for the auxotrophic factor and the therapeutic factor. A modified host cell capable of expressing. 33. The modified host cell of claim 33, which is a mammalian cell. 33. The modified host cell of claim 33, which is a human cell. Embryonic stem cells, stem cells, precursor cells, pluripotent stem cells, artificial pluripotent stem (iPS) cells, somatic stem cells, differentiated cells, mesenchymal stem cells, neural stem cells, hematopoietic stem cells or hematopoietic precursor cells, adipose stem cells, keratinocytes 33. The modified host cell according to claim 33, which is selected from the group consisting of skeletal stem cells, muscle stem cells, fibroblasts, NK cells, B cells, T cells, and peripheral blood mononuclear cells (PBMC). 33. The modified host cell of claim 33, which is derived from a cell from a subject treated with the modified host cell. A method for producing modified mammalian host cells.
(a) Nucleic acids encoding one or more nuclease systems that target and cleave the DNA in the nutritionally demanding induction locus, or one or more components of the one or more nuclease system. Introducing into the mammalian host cell, and
(b) said method comprising the donor template or vector according to any one of claims 1-28. A second nuclease or a second guide RNA for targeting and cleavage of the DNA at the second genomic locus, or a nucleic acid encoding the second nuclease or the second guide RNA, and optionally.
(b) The method of claim 38, further comprising introducing a second donor template or vector. A method of targeting the integration of an introductory gene into a feeding-requiring induction locus of a mammalian cell in Exvivo, wherein the mammalian cell is contacted with the donor template or vector and nuclease according to any one of claims 1-28. The above-mentioned method, which comprises causing. The method of any one of claims 38-40, wherein the nuclease is a ZFN. The method according to any one of claims 38 to 40, wherein the nuclease is TALEN. A method for producing modified mammalian host cells.
(a) Cas9 polypeptide or nucleic acid encoding the Cas9 polypeptide,
(b) Guide RNA specific for the auxotrophic induction locus, or nucleic acid encoding the guide RNA, and
(c) The method comprising introducing the donor template or vector according to any one of claims 1-28 into the mammalian host cell. (a) A second guide RNA specific for the second auxotrophic induction locus, or a nucleic acid encoding the guide RNA, and optionally.
(B) The method of claim 42, further comprising introducing a second donor template or vector into said mammalian host cell. With the donor template or vector, Cas9 polypeptide, and guide RNA according to any one of claims 1-28, which is a method for targeting the integration of an introductory gene into a feeding-requiring induction locus of a mammalian cell in Exvivo. The method comprising contacting the mammalian cells. The method according to any one of claims 43 to 45, wherein the guide RNA is a chimeric RNA. The method of any one of claims 43-45, wherein the guide RNA comprises two hybridized RNAs. The method of any one of claims 38-45, further comprising causing one or more single-strand breaks within the auxotrophic lotus coition. The method of any one of claims 38-45, further comprising causing double-strand breaks within the auxotrophic lotus coition. The method of any one of claims 38-49, wherein the auxotrophic induction lous coition is modified by homologous recombination using the donor template or vector. The method according to any one of claims 38 to 49, wherein steps (a) and (b) are performed before or after the expansion of the cells and optionally the culture of the cells. (c) The method of claim 51, further comprising selecting cells containing the transgene integrated into the auxotrophic induction locus. The above choice is
(i) Select cells that require the auxotrophy factor to survive; and optionally.
(ii) The method of claim 52, comprising selecting cells containing the transgene integrated into the auxotrophic induction locus. 52. The method of claim 52, wherein the auxotrophic induction locus is the gene encoding uridine monophosphate synthase and the cells are selected by contact with 5-FOA. Aseptic composition comprising the donor template or vector according to any one of claims 1-28 or the nuclease system according to any one of claims 29-32, and sterile water or pharmaceutically acceptable excipients. thing. A sterile composition comprising the modified mammalian host cell of any one of claims 33-37 and sterile water or a pharmaceutically acceptable excipient. The donor template or vector according to any one of claims 1 to 28 , the nuclease system according to any one of claims 29 to 32 , or the modified host cell according to any one of claims 33 to 37 , or A kit containing these combinations, optionally with a container or vial. A pharmaceutical for use in a method of treating a subject having a disease, disorder, or condition, which comprises (a) the modified host cell and (b) the nutritional requirement factor, wherein the method is a therapeutic amount. The modified host cell according to any one of claims 33 to 37 and (b) the nutritionally demanding factor are administered to the subject in an amount sufficient to bring about the expression of the therapeutic factor. The above-mentioned medicine including the above-mentioned medicine . The disease, the disorder, or the condition is cancer, Parkinson's disease, graft-versus-host disease (GvHD), autoimmune condition, hyperproliferative disorder or condition, malignant transformation, liver condition, hereditary genetic defect. 58. The pharmaceutical agent according to claim 58 , which is selected from the group consisting of a genetic condition including, juvenile diabetes, and an eye compartment condition. The disease, the disorder, or the condition is selected from the group consisting of the muscular system, the skeletal system, the circulatory system, the nervous system, the lymphatic system, the respiratory system, the endocrine system, the digestive system, the excretory system, and the reproductive system. The pharmaceutical according to claim 59 , which affects at least one system of the body. Pharmaceutically according to any one of claims 33 to 37, comprising the modified host cell for use in human administration or in the treatment of a disease, disorder, or condition. A pharmaceutical agent comprising an auxotrophic factor for use in administration of the modified human host cell according to any one of claims 33 to 37 to a given human. A drug for use in a method of alleviating or treating a disease or disorder in a subject in need thereof .
(a) A composition comprising a modified host cell containing a transgene encoding a protein integrated into an auxotrophic induction locus, wherein the modified host cell is auxotrophic for a trophic factor; and
(b) Combining sufficient amounts of auxotrophic factors to result in therapeutic expression of the protein.
The method comprises administering the composition and the auxotrophic factor to the subject.
The drug . The pharmaceutical according to claim 63 , wherein the auxotrophic induction locus is in a gene encoding uridine monophosphate synthase (UMPS). The medicine according to claim 64 , wherein the auxotrophic factor is uridine. The pharmaceutical according to claim 63 , wherein the auxotrophy-inducing lous coition is within a gene encoding holocarboxylase synthase (HLCS). The medicine according to claim 66 , wherein the auxotrophic factor is biotin. The medicine according to claim 63 , wherein the protein is an enzyme. The medicine according to claim 63 , wherein the protein is an antibody. The modified host cells include embryonic stem cells, stem cells, precursor cells, pluripotent stem cells, artificial pluripotent stem (iPS) cells, somatic stem cells, differentiated cells, mesenchymal stem cells, nerve stem cells, hematopoietic stem cells or hematopoietic precursors. 39. The pharmaceutical according to claim 63 , which is a cell, adipose stem cell, keratinocyte, skeletal stem cell, muscle stem cell, fibroblast, NK cell, B cell, T cell, or peripheral blood mononuclear cell (PBMC). The medicine according to claim 63 , wherein the modified host cell is a mammalian cell. The medicine according to claim 71 , wherein the mammalian cell is a human cell. The medicine according to claim 63 , wherein the modified host cell is derived from the subject in which the modified host cell is treated with the modified host cell. The medicine according to claim 63 , wherein the composition and the auxotrophic factor are administered at the same time. The medicine according to claim 63 , wherein the composition and the auxotrophic factor are continuously administered. The pharmaceutical according to claim 75 , wherein the composition is administered prior to the auxotrophic factor. The medicine according to claim 63 , wherein the composition and the auxotrophic factor are administered at the same time. 13. The apparatus of claim 63 , wherein administration of the auxotrophic factor is continued periodically for a period sufficient to promote therapeutic expression of the protein. 33. The apparatus of claim 63 , wherein the administration of the auxotrophic factor is reduced in order to reduce the expression of the protein. 33. The apparatus of claim 63 , wherein the administration of the auxotrophic factor is increased in order to increase the expression of the protein. 13. The apparatus according to claim 63 , wherein discontinuation of administration of the auxotrophic factor induces proliferation inhibition or cell death of the modified host cell. 13. The apparatus according to claim 63 , wherein administration of the auxotrophic factor is continued for a period sufficient to exert a therapeutic effect in the subject. The medicine according to claim 63 , wherein the modified host cell is regenerative. 63. The apparatus of claim 63 , wherein administration of the composition comprises topical delivery. 33. The apparatus of claim 63 , wherein administration of the auxotrophic factor comprises systemic delivery. The apparatus according to claim 63 , wherein the disease is lysosomal storage disease (LSD). The diseases are Gauche disease (type 1/2/3), MPS2 (hunter) disease, Pompe disease, Fabry disease, Clave disease, hypophosphatase disease, Niemann-Pick disease type A / B, MPS1, MPS3A, MPS3B, MPS3C. , MPS3, MPS4, MPS6, MPS7, phenylketonuria, MLD, Sandhoff's disease, Teisax 's disease, and Batten's disease .
The proteins are glucocerebrosidase, idulsulfase, alglucosidase alpha, agarsidase alpha, agarsidase beta, galactosylceramidase, ashotase alpha, acidic sphingomyelinase, laronidase, heparan N-sulfatase, alpha-N-. Acetylglucosaminidase, heparan-α-glucosaminide N-acetyltransferase, N-acetylglucosamine 6-sulfatase, erosulfase alpha, glasulfate, B-glucuronidase, phenylalanine hydroxylase, arylsulfatase A, hexosaminidase-B, hex The pharmaceutical according to claim 63 , which is an enzyme selected from the group consisting of sosaminidase-A and tripeptidyl peptidase 1. 33. The apparatus of claim 63 , wherein the disease is Friedreich ataxia, hereditary angioedema, or spinal muscular atrophy and the protein is frataxin, C1 esterase inhibitor, or SMN1 . A pharmaceutical agent comprising the modified host cell according to any one of claims 33 to 37, for reducing the size of a tumor or slowing the rate of tumor growth in a subject.